Clinical and Vaccine Immunology, 2015, January; 22(1):56-64.

A Eukaryotic Expression Plasmid Carrying Chicken Interleukin-18 Enhances the Response to Newcastle Disease Virus Vaccine

Chen Wang, Xiaokang Li, Chunjie Zhang, Tingcai Wu, Yinju Li, Xiangchao Cheng

College of Animal Science and Technology, Henan University of Science and Technology, Luoyang, China

 

Abstract

Interleukin-18 (IL-18) is an important cytokine involved in innate and acquired immunity. In this study, we cloned full-length chicken IL-18 (ChIL-18) gene from SPF chicken embryo spleen cells and provided evidence that ChIL-18 gene in recombinant plasmid was successfully expressed in chicken DT40 cell. ChIL18 significantly enhanced interferon-γ mRNA expression in chicken splenocytes, which increased interferon-γ-induced nitric oxide (NO) synthesis by macrophages. The potential genetic adjuvant activity of ChIL-18 plasmid was examined in chickens by co-injection of ChIL-18 plasmid and Newcastle disease (ND) inactivated vaccine. ChIL-18 markedly elevated serum hemagglutination inhibition (HI) titers and anti-hemagglutinin-neuraminidase (anti-HN) specific antibody levels, induced the secretion of both Th1 (interferon-γ) and Th2 (interleukin-4) type cytokines, promoted the proliferation of T and B lymphocytes, and increased populations of CD3+T cells and their subsets, CD3+CD4+ and CD3+CD8+ T cells. Furthermore, virus challenge revealed that ChIL-18 contributed to protection against Newcastle disease virus challenge. Taken together, our data indicate that the co-administration of ChIL-18 plasmid and ND vaccine induces a strong immune response at both the humoral and cellular levels and that ChIL-18 is a novel immunoadjuvant suitable for ND vaccination. 

PMID: 25355794

 

Supplement: 

The isolation and characterization of chicken IL-18 (ChIL-18) was reported in 2000 by Schneider et al. (1), and when expressed in Escherichia coli could induce interferon (IFN)-γ production. Thomas et al. (2) reported that ChIL-18 has many biological effects, including the activation and proliferation of Th cells, and inducing CD4+ T cells to secrete IFN-γ. Hung et al. (3) confirm that chicken IL-18 recombinant proteins exert in vivo biological functions through stimulating humoral and cell-mediated immunities in order to enhance the antigen-specific immunity and vaccine efficacy. Although many studies have shown recombinant ChIL-18 boosts the immune responses of avian vaccines (4-6), However, few studies have investigated the modulatory effect of using a eukaryotic expression plasmid carrying ChIL-18 as a molecular genetic adjuvant to enhance avian vaccines. In this study, we cloned full-length ChIL-18 gene from SPF chicken embryo spleen cells and report a eukaryotic expression plasmid carrying ChIL-18 as a genetic adjuvant, co-administrated with ND inactivated vaccine. We hypothesized that ChIL-18 as a genetic adjuvant, co-administrated with ND inactivated vaccine, induce a strong immune response in chickens at both the humoral and cellular levels. 

Many studies have shown ChIL-18 express in non-chicken derived cell (3). However, there are no documents whether the ChIL-18 express IL-18 in chicken cells or not. To address this, the expression analysis of the recombinant pcDNA3.1/His-ChIL-18 plasmid was performed in chicken DT40 cells. The DT40 chicken B cell line is derived from an avian leukosis virus (ALV)-induced bursal lymphoma. Moreover, it is arrested at a bursal stem cell stage of differentiation, suggesting it may be best characterized as being a bursal pre-B cell line (7). We found that the protein ChIL-18 specifically recognized by anti-His-tag monoclonal antibody in a dose-dependent manner. The purified ChIL-18 protein has a major band with a Mr of approximately 25,000 and specific probe by anti-His Tag monoclonal antibody. Therefore, this paper provided evidence that ChIL-18 gene in recombinant plasmid was successfully expressed in chicken DT40 cell. For further evaluate bioassays of the full-length ChIL-18 gene expressed in eukaryotic cell, a sensitive bioassay for ChIL-18 in vitro based on the inducible release of IFN- γ was performed. We found that the ChIL-18 protein expressed in eukaryotic DT40 cell increased significantly enhanced IFN- g mRNA expression in chicken splenocytes, which increased interferon-γ-induced nitric oxide (NO) synthesis by macrophages. These results show that the amino acid mutation (Ser to Phe) in the ChIL-18 protein does not modify the activity of IL-18.

Cytokines play an important role in development of the immune system and in responses to infection (8). IL-18 is a strong IFN-γ inducing factor that enhances immune responses as an adjuvant. Currently, the concept of using IL-18 to boost immune responses after vaccination has been investigated for chicken vaccines (5,6) .Hung et al.(3) described that full length and mature chicken IL-18 purified recombinant protein effectively enhanced cell-mediated and humoral immunity. However, the short half-life of recombinant cytokines and side effects due to repetitive administration are major disadvantages for their use (9). Previous reports showed that direct injection of cytokine genes into muscles could modulate immune responses (10). Here, we report a eukaryotic expression plasmid carrying ChIL-18, to investigate its immunogenic potential as a genetic adjuvant to induce immune responses in chickens. Upon ND vaccination, the ChIL-18 plasmid stimulated significant antigen-specific immune responses. The level of HI antibodies present 7 days following co-inoculation of ChIL-18 eukaryotic expression plasmid and ND vaccine was higher than that induced by inoculation with the vaccine alone, and this enhancement was still detectable up to 21 days post-inoculation. Moreover, ChIL-18 promoted T and B lymphocyte proliferation responses, which were higher than in the group inoculated with vaccine alone on day 7 and 21 after the first vaccination. These data indicate that ChIL-18 induces a strong immune response at both the humoral and cellular levels.

The importance of this study is two-fold.  First, this paper provided evidence that ChIL-18 gene in recombinant plasmid was successfully expressed in chicken DT40 cell. We found that the ChIL-18 protein expressed in eukaryotic DT40 cell increased significantly enhanced IFN-γ mRNA expression in chicken splenocytes, which increased interferon-γ-induced nitric oxide (NO) synthesis by macrophages. Second, this study indicates that the co-administration of ChIL-18 plasmid and ND vaccine induces a strong immune response at both the humoral and cellular levels and suggests ChIL-18 is a novel immunoadjuvant suitable for ND vaccine.

 

References 

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Acknowledgements 

This work was supported by the grant No.31101792 and No.31201928 from the National Natural Science Foundation of China and No. 2012GGJS-077 from the Foundation for University Key Teacher by Higher Education of Henan Province.

 

Contact:

Dr. Chen Wang

College of Animal Science and Technology,

Henan University of Science and Technology,

Luoyang, Henan, China

Tel: +86 379 65507210;Fax: +86 379 64282341

E-mail address: wangchen2001@ 126.com

 

 

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